One of the puzzles in cancer predisposition is that women carrying BRCA-1 mutations preferentially develop tumors in epithelial tissues of the breast and ovary. Moreover, sporadic breast tumors contain lower levels of BRCA-1 in the absence of mutations in the BRCA-1 gene. The problem of tissue specificity requires analysis of factors that are unique to tissues of the breast. For example, the expression of estrogen receptor-alpha (ER alpha) is inversely correlated with breast cancer risk, and 90% of BRCA-1 tumors are negative for ER alpha. Here, we show that estrogen stimulates BRCA-1 promoter activity in transfected cells and the recruitment of ER alpha and its cofactor p300 to an AP-1 site that binds Jun/Fos transcription factors. The recruitment of ER alpha/p300 coincides with accumulation in the S-phase of the cell cycle and is antagonized by the antiestrogen tamoxifen. Conversely, we document that overexpression of wild-type p53 prevents the recruitment of ER alpha to the AP-1 site and represses BRCA-1 promoter activity. Taken together, our findings support a model in which an ER alpha/AP-1 complex modulates BRCA-1 transcription under conditions of estrogen stimulation. Conversely, the formation of this transcription complex is abrogated in cells overexpressing p53.

Previous reports have documented the antiproliferative properties of a mixture of conjugated isomers (CLA) of linoleic acid [LA (18:2)]. In this study, we investigated the mechanisms of CLA action on cell cycle progression in breast and colon cancer cells. Treatment with CLA inhibited cell proliferation in breast cancer MCF-7 cells containing wild-type p53 (p53(+/+)). At cytostatic concentrations, CLA elicited cell cycle arrest in G1 and induced the accumulation of the tumor suppressors p53, p27 and p21 protein. Conversely, CLA reduced the expression of factors required for G1 to S-phase transition including cyclins D1 and E, and hyperphoshorylated retinoblastoma Rb protein. In contrast, the overexpression of mutant p53 (175Arg to His) in MFC-7 cells prevented the CLA-dependent accumulation of p21 and the reduction of cyclin E levels suggesting that the expression of wild-type p53 is required for CLA-mediated activation of the G1 restriction point. To further elucidate the role of p53, the effects of CLA in colon cancer HCT116 cells (p53(+/+)) and p53-deficient (p53(-/-)) HCT116 cells (HCTKO) were examined. The treatment of HCT116 cells with CLA increased the levels of p53, p21, p27 and hypophosphorylated (pRb) protein and reduced the expression of cyclin E, whereas these effects were not seen in p53-deficient HCTKO cells. The t10,c12-CLA isomer was more effective than c9,t11-CLA in inhibiting cell proliferation of MCF-7 breast cancer cells and enhancing the accumulation of p53 and pRb. We conclude that the antiproliferative properties of CLA appear to be a function, at least in part, of the relative content of specific isomers and their ability to elicit a p53 response that leads to the accumulation of pRb and cell growth arrest.

The effects of a ligand of the aromatic hydrocarbon receptor (AhR), benzo[a] pyrene (B[ a]P), and its metabolite, BPDE (7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene), on BRCA-1 levels and cell cycle kinetics were determined in MCF-7 breast cancer cells. Exposure of asynchronous MCF-7 cells for 72 hours to a non-cytotoxic dose of 0.5 microM B[a]P triggered a three-fold reduction in BRCA-1 protein. In MCF-7 cells resistant (20% to 30%) to genotoxic concentrations of B[a]P (1 to 5 microM), the loss of BRCA-1 protein was coupled with pausing in S-phase and G2/M, and accumulation of p53, mdm2 and p21. Treatment of MCF-7 cells synchronized in S-phase (72%) with B[a]P prolonged the arrest in S-phase, although this checkpoint was transient since cells resumed to G2/M after 12 hours with reduced levels of BRCA-1. In these cells, levels of p53 were increased, whereas the cellular content of p21 remained unaltered. In contrast, the co-treatment with the AhR antagonist, alpha-naphthoflavone (ANF), abrogated the deleterious effects of B[a]P on BRCA-1 expression, while preventing the accumulation of p53 and disruption of cell cycle profile. These findings suggest that the AhR mediated the inverse expression patterns of BRCA-1 and p53 upon exposure to B[a]P. The treatment with BPDE induced S-phase arrest and reduced BRCA-1 mRNA levels. The negative effects of BPDE on BRCA-1 expression were not transient since removal of BPDE did not allow complete reversal of the repression. These cumulative data suggest that the B[a]P metabolite, BPDE, may play a key role in disruption of BRCA-1 expression and cell cycle kinetics in breast epithelial cells.

In the absence of a causal relationship between the incidence of sporadic breast cancer and occurrence of mutations in breast cancer susceptibility genes, efforts directed to investigating the contribution of environmental xenobiotics in the etiology of sporadic mammary neoplasia are warranted. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants, which have been shown to induce DNA damage and disrupt cell cycle progression. In this report we discuss published data pointing to PAHs as a risk factor in carcinogenesis, and present findings generated in our laboratory suggesting that the mammary tumorigenicity of PAHs may be attributable, at least in part, to disruption of BRCA-1 expression by reactive PAHmetabolites. We report that benzo[a]pyrene (B[a]P), selected as a prototype PAH, disrupts BRCA-1 transcription in estrogen receptor (ER)-positive but not ER-negative breast cancer cells. The reduced potential for BRCA-1 expression in B[a]Ptreated cells coincides with disruption of cell cycle kinetics and accumulation of p53. These effects are counteracted by the AhR-antagonist ␣-naphthoflavone (ANF), and in breast cancer cells expressing mutant p53 or the E6 human papilloma virus protein. We suggest that exposure to PAHs may be a predisposing factor in the etiology of sporadic breast cancer by disrupting the expression of BRCA-1. Environ. Mol. Mutagen. 39:235-244, 2002.

The objective of this study was to investigate whether polycyclic aromatic hydrocarbons (PAHs) contribute to the etiology of sporadic breast cancer by altering the expression of BRCA-1. Acute exposure to the PAH benzo[a]pyrene (B[a]P) inhibited in a time- and dose-dependent fashion cell proliferation and levels of BRCA-1 mRNA and protein in estrogen receptor (ER)-positive breast MCF-7 and ovarian BG-1 cancer cells. Moreover, the acute exposure to B[a]P abrogated estrogen induction of BRCA-1 in MCF-7 cells. The loss of BRCA-1 expression was prevented by the aromatic hydrocarbon receptor (AhR) antagonist alpha-naphthoflavone, suggesting participation of the AhR pathway. BRCA-1 exon 1a transcripts were downregulated by B[a]P faster than exon 1b mRNA was. Long-term exposure to B[a]P (40 nM for 15 mo) lowered BRCA-1 mRNA levels in subclones of MCF-7 and BG-1 cells, whereas expression of BRCA-1 in these clones was reverted to normal levels by washing out of B[a]P. The mechanisms of BRCA-1 repression by B[a]P were further investigated by examining the effects of the halogenated aryl hydrocarbon 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and the B[a]P metabolite 7r, 8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). While TCDD did not influence basal BRCA-1 mRNA and protein levels at any of the doses (from 10 nM to 1 microM) tested in this study, treatment with 50 nM BPDE drastically reduced BRCA-1 mRNA levels, indicating that metabolism of B[a]P to BPDE may contribute to downregulation of BRCA-1. Conversely, ER-negative breast MDA-MB-231 and HBL-100 cancer cells were refractory to treatment with B[a]P or TCDD and expressed constant levels of BRCA-1 mRNA and protein. We conclude that B[a]P may be a risk factor in the etiology of sporadic breast cancer.